Pneumocystis (PC) is a dimorphic fungus that causes pneumonia in immunocompromised individuals and PC pneumonia (PCP) is the most common serious opportunistic infection in individuals diagnosed with HIV. Furthermore, PCP is re-emerging in the clinical setting of non-HIV infected immunocompromised individuals receiving life-saving immunosuppressive therapies for conditions such as hematological malignancies, transplantation, and autoimmune conditions. As the at-risk population continues to increase, finding novel, conserved, extracellular antigens on PC is the first step towards designing more effective diagnostic tests, creating monoclonal antibody based therapies, and developing vaccines. To discover novel antigens, proteomic and transcriptome analysis of PC was conducted. Specifically, surface biotinylation of total PC, containing both the cyst ant troph life form, was performed and demonstrated that histone 2B (H2B) was an abundant extracellular protein on PC. RNA sequencing was also performed on separated PC cysts and trophs to identify targets differentially expressed in the two life forms. H2B had a 3-fold increase in expression in the cyst form when compared to the troph form, while other proteins, such as a Meu10, were found to have higher expression in the troph form. To determine the immunogenicity of H2B, mice were vaccinated with an H2B DNA vaccine. Mice receiving the H2B vaccine had a significant increase in anti-PC serum IgG and had a significant decrease in lung PC burden when compared to unvaccinated controls. These experiments demonstrate that H2B is a cyst-enriched antigen and is capable of providing protection against developing PCP following DNA vaccination. The aims of this proposed project are: 1.) To define the humoral immune response generated by H2B DNA vaccination, and 2.) To determine the immunogenicity of a Meu10 DNA vaccine and evaluate the protection provided by a life cycle based multivalent DNA vaccination. To accomplish these aims, the proposed studies will explore the memory B cell response generated by H2B DNA vaccination and examine the immunologic mechanisms of protection provided by the vaccine. Furthermore, vaccination studies will be conducted with a Meu10 DNA vaccine and a multivalent vaccine targeting both the cyst-enriched H2B and the troph-enriched Meu10 to examine the efficacy of a targeted, life cycle based multivalent vaccine. The proposed studies will define the fundamental immunological mechanisms behind a previously designed H2B vaccine and will explore a novel strategy for vaccine development for the re- emerging infectious disease of PC in accordance with the mission statement of the NIAID.